Major obstacles in the development of HIV vaccine have been identifying a prophylactic strategy that affords protection against the growing number of HIV subtypes and strains and determining the immune correlates of protection. To this end, our laboratory has investigated the inactivated virus vaccine approach using the feline immunodeficiency virus (FIV)-cat model of AIDS. Protection against homologous and closely related FIV strains has been achieved with single-strain vaccines, whereas protection against heterologous subtypes and even distinctly heterologous strains of the same subtype has been difficult. In order to broaden the protective immunity, FIV strains from different subtypes have been combined as immunogens of dual- and triple-subtype FIV vaccines. Preliminary results suggest that at a set antigenic dose, the triple-subtype vaccine was not as effective as the dual-subtype vaccine in protecting cats against heterologous strains of same and different subtypes. Furthermore, the dual-subtype vaccine was more effective at eliciting the appropriate, reproducible immune responses in the vaccinated cats with the detection of virus neutralizing antibodies, cytotoxic T Iymphocyte activities, and T-helper (TH) activities including vaccine-induced TH-I cytokines such as interferon-gamma. Controversy exists over what types of immunity are essential for prophylactic protection against AIDS viruses and the types of challenge systems to use such as intravenous versus mucosal challenge and in vivo-derived versus in vitro-derived inoculum. In effort to address these issues, antibody-free immunocytes from vaccinated cats will be adoptively transferred to cats with matched-immune system. Cats with matched-immune systems were developed by bone marrow transplantation. In a recent preliminary study, recipients of the adoptive transfer immunocytes were protected against homologous FIV challenge. In this competitive renewal grant, the correlate of protective immunity induced by dual-subtype FIV vaccine will be identified by performing adoptive transfer studies on cats with matched immune systems. Our goal is to determine the cellular immune mechanisms of dual-subtype vaccine protection against homologous and heterologous subtype challenges and to test whether such protection is effective against mucosal/vaginal challenge.